Reciprocal impacts of telomerase activity and ADRN/MES differentiation state in neuroblastoma tumor biology

Telomere maintenance and tumor cell differentiation have been separately investigated in neuroblastoma malignancy. Their mechanistic connection is unclear. We establish a tight and reciprocal causal relationship between the telomere and differentiation programs of high-risk neuroblastoma.

Neuroblastoma is the most prevalent extra-cranial solid tumor affecting children and accounts for ~10% of all pediatric cancer mortality 1. It’s best known for its unique and remarkable heterogeneity: depending on the genetic make-up of the tumor, the outcomes for the patient can range from spontaneous regression to lethal metastatic disease resistant to all therapy 2, 3, 4. Despite modern combinations of surgery, radiation, chemotherapy and immunotherapy, nearly 50% of high-risk neuroblastoma remains incurable 5. New mechanism-based strategies that target critical pathways constitute major unmet needs.

High-risk neuroblastoma typically carries a set of recurrent genetic aberrations including MYCN amplification, TERT promoter rearrangement, and ATRX mutation. Recent studies indicate that each of these aberrations is connected to the activation of a telomere maintenance mechanism (TMM), i.e., telomerase or ALT (alternative lengthening of telomeres) 6. This is not particularly surprising given that telomeres have a critical role in sustaining the proliferative capacity of tumor cells. Indeed, for most cancer types, tumor samples almost invariably harbor either telomerase or ALT. More surprising is the fact that many neuroblastoma patients can present with sizable tumors with metastatic spread that harbor no telomere maintenance mechanism, and these are the tumors that spontaneously regress. While the underlying reason has not been established, a plausible explanation is that because neuroblastoma originates from neural crest progenitor cells (more specifically progenitor cells of the sympathoadrenal lineage) that likely harbor very long telomeres, even tumors without TMMs may have sufficient telomere reserve to support many rounds of cell division 7. Therefore, neuroblastoma is able to provide a compelling illustration of the critical importance of telomere maintenance for tumor cells, i.e., tumors with active TMM are highly malignant, and those without TMM can be cured with time 8.

Recent studies also highlight another facet of neuroblastoma that can be attributed to its unique developmental origin, namely tumor cell heterogeneity based on cell lineage and differentiation. While this heterogeneity was discovered more than three decades ago – through the pioneering studies of June Biedler and colleagues 9, recent transcriptomic and epigenetic profiling have provided detailed insights on the molecular differences between distinct cell types, which are now classified as ADRN (adrenergic) or MES (mesenchymal) 10. Importantly, these two cell types are interconvertible and have been implicated in disease progression as well as treatment response. For example, the MES cells are more chemo-resistant and may be enriched in relapse and in metastatic diseases 10, 11. In contrast, the ADRN cells tend to predominate at diagnosis and show greater sensitivity to chemotherapies 10, 12.  Understanding the role of tumor cell differentiation in high-risk neuroblastoma malignancy is therefore crucial for developing new and effective therapies.

Despite strong interests in both telomeres and tumor cell differentiation, few studies have addressed the potential connections between these two critical facets of neuroblastoma tumor biology. This knowledge gap was one of the key reasons that motivated our investigation. Previously, my lab (Lue lab) had primarily studied telomere functions and telomere maintenance mechanisms in model organisms. Being able to team up with the Nai-Kong Cheung lab at Memorial Sloan Kettering Cancer Center (MSK) was another motivating factor and a timely development. As the busiest referral center in the world, the MSK neuroblastoma service offers a treasure trove of archived samples linked to clinical information for both biological and therapeutic discoveries.

In our study 13, we set out to address the roles of telomere-related factors in the different lineages of neuroblastoma tumor cells. Using isogenic cell lines that display either ADRN or MES phenotypes, we showed that the levels of telomere factors vary dramatically, with the ADRN cells generally harboring much higher levels of such factors (e.g., key components of the shelterin complex TRF1 and TRF2). Pharmacologically converting ADRN cells into MES cells triggered the expected changes in telomere protein profiles. Conversely, inhibiting telomerase activity in ADRN cells induced their conversion into MES cells in a reversible manner. Together, these findings establish for the first time a tight mechanistic connection between telomere regulation and neuroblastoma lineage conversion, and suggest that inhibiting telomerase may alter malignancy not only by reducing proliferation but also by changing the differentiation status of tumor cells. In the course of our study, we discovered a strong up-regulation of DNA-sensing and innate immunity factors in MES cells, which resulted in substantially greater immunogenicity for these cells. Hence the connection between telomeres and tumor cell lineage is linked to yet another facet of neuroblastoma with therapeutic implications.

 

Figure. Telomeres and neuroblastoma ADRN/MES differentiation

 

Are these findings relevant to patients’ tumors? We examined this question by analyzing the transcription profiles of neuroblastoma tumor samples in the publicly available databases. Using cluster analysis and a gene expression signature that incorporates telomere- and cell lineage-related factors, my colleague Xi Zhou (Weill Cornell) was able to segregate patient samples into predominantly ADRN or MES-like groups and show that these groups manifest distinct survival outcomes. We therefore surmise that tumors with predominantly ADRN or MES cells do exist in vivo and that the telomere and lineage characteristics of the tumors affect their clinical behaviors.

In summary, for telomerase-positive neuroblastoma, we have established a set of mechanistic connections between telomere regulation, tumor cell differentiation and tumor cell immunogenicity. Our findings suggest that eliminating telomere maintenance may tackle three major hallmarks of neuroblastoma malignancy, i.e., dysregulated proliferation, differentiation, and immune evasion. Inhibiting telomerase may prove to be especially efficacious against high-risk neuroblastoma – by simultaneously eliminating telomere maintenance and enhancing immunogenicity through tumor cell lineage reprogramming.

This project was spearheaded by my colleague Eun Young Yu, who introduced and developed many techniques that are new to the lab. She received strong assistance from two other lab members, Syed Zahid and Sarah Aloe.  We also benefited greatly from our collaboration with Nai-Kong Cheung and Xi Zhou. Having made the interesting discoveries for telomerase-positive neuroblastoma, Eun Young is now turning her attention to the ALT-positive subgroup of cancers, which has a very different clinical presentation and is characterized by different genetic aberrations. We anticipate that investigating the relationship of telomeres to differentiation and immunogenicity in this subgroup will also be very informative.

  

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